Abstract 5119: Quantitative proteomics of myeloid cells reveal upregulation of the Fc receptor and integrin pathways in a mouse ovarian cancer model

Abstract

Abstract The tumor microenvironment exerts a profound effect on tumor growth and therapeutic response. Innate immune bone marrow derived cells (BMDCs), including tumor-associated macrophages, and myeloid-derived suppressor cells are prominent in cancer hosts, and their presence correlates with poor prognosis for many human cancers. Here, we examine the role of BMDCs in a mouse model for advanced ovarian cancer. Ovarian cancer is the 5th leading cause of cancer-related deaths in women. Our model uses ovarian epithelial cancer cell lines transplanted into the peritoneum of C57BL/6 mice. These mice develop malignant ascites by 10 weeks, with seeding of tumor nodules throughout the peritoneum. Peritoneal macrophages were flow sorted for high levels of CD11b and further gated on MHC class II to yield CD11b high MHCII+ (MHC+) and CD11b high MHCII low (MHClow) subsets. MHC+ cells in the CD11b high fraction increased from 18% in naïve mice to 32% in tumor bearing mice. Myeloid cells from spleens were flow sorted based on expression of CD11b, Ly6C and Ly6G to yield CD11b high Ly6C+Ly6G- (Ly6C+) and CD11b high Ly6Cint Ly6G+(Ly6G+) subsets with monocytic and granulocytic morphology respectively. Ly6C+ but not Ly6G+ cells respond to GM-CSF with colony formation at a frequency of 1 in 100 input cells. 30-50% of cells from the CFUs were CD11b+CD11c+, consistent with the presence of dendritic cell precursors. Although there was a trend towards increased frequency of CFUs in Ly6C+ cells from tumor bearing mice, the difference was not significant. Using quantitative mass spectrometry (QMS), we analyzed protein expression in these four subsets. Tissue from 24 tumor bearing mice and 14 groups of control mice were analyzed, generating 117 cellular populations subject to QMS using an accurate mass time tag (AMT) strategy on an LTQ Orbitrap XL mass spectrometer. In 277 runs, we identified 3,692 proteins with 35,552 AMT tags. Protein expression levels were based on 5.39 million MS1 ion current measurements. Comparing samples from tumor bearing animals to controls, pathway and network analyses revealed extensive changes in the cellular biochemistry of all populations. Both the MHC+ and Ly6C+ subsets show a notable increase in the level of expression of proteins in integrin and integrin linked kinase signaling (extracellular matrix interactions) and Fc γ receptor pathways (cytokine secretion, M2 polarization). Key molecules increasing in expression include integrins β 2, 3 and 5, parvin b, PKC α and β and PAK kinases. These pathways are down regulated or show mixed changes in MHC- and Ly6G+ cells. Clathrin mediated endocytic pathways are upregulated in all subsets from tumor bearing mice, consistent with dysregulated cellular trafficking. Our findings identify many novel candidates for drug targeting and therapeutic intervention. This work was supported in part by NIH R01 CA85368 to A. W. Lee. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5119. doi:10.1158/1538-7445.AM2011-5119